Food and water-borne bacterial pathogens remain a major global threat to public health. The emergence of multidrug-resistant strains of bacteria and new pathogens require the development of new approaches to antibiotic discovery. Genetic, cellular and biochemical studies of virulence mechanisms have revealed protein translocation by type III secretion systems (T3SSs) as a key pathway for disarming host defenses during Gram-negative bacterial infection. Bacterial mutants with defective T3SSs cannot inject effector proteins into host cells and are less capable of causing disease in animal models. Components of the T3SS apparatus are therefore excellent antibiotic targets. This project will focus on the development of T3SS inhibitors targeted at infections caused by Salmonella. Strains of this intracellular bacterial pathogen are one of the leading causes of gastroenteritis and typhoid fever in humans. To identify antibacterial drugs that effectively targe T3SSs and control bacterial infection, this research application aims to 1) develop potent and selective T3SS inhibitors, 2) construct and characterize photoaffinity and Click-able probes based on these inhibitors and 3) determine their specific targets and mechanism of action. The goal of this research is creation of extremely potent T3SS inhibitors and elucidating of the working mechanism of their inhibition. Knowledge of the specific interactions and mechanism of infection is often the bottle-neck step of drug development. The results of these non-trivial experiments will be specific advances in the study of T3SS inhibitors, and a better general understanding of compounds that inhibit bacterial infection. These results are expected to extend beyond Salmonella infection. Since many other bacterial pathogens utilize the highly conserved type III secretion machinery, the discovery and characterization of more potent and specific inhibitors of Salmonella secretion will likely contribute to the identification of a new cass of drugs effective against a wide range of bacterial pathogens. PUBLIC HEALTH RELEVANCE: This research will focus on the development of type III secretion system (T3SS) inhibitors targeted at infections caused by Salmonella enterica, a Gram-negative bacterial pathogen, that is one of the leading causes of food-borne illness in the USA and responsible for typhoid fever world-wide. Because T3SSs are conserved in many bacterial pathogens, the discovery and characterization of more potent inhibitors of secretion in Salmonella will contribute to the development of antibacterial drugs effective against other enteric pathogens.